The "therapeutic index" of a drug is defined as the "ratio between a lethal and an effective therapeutic dose". For insulin this index is extremely low (Brange, Y., in Galenics of Insulin; Springer-Verlag, N.Y., 1987). For this reason, insulin is a dangerous drug. The clinical consequence of overtreatment is coma or death. Exacerbating this delicate clinical picture is the substantial day to day variation in the rate and duration of the subcutaneous absorption of insulin (Schlichtkrull, J. et al., Handbook of Experimental Pharmacology, Hasselblatt A. (ed), vol XXXII/2,1975, Springer-Verlag, N.Y.) and this is a major cause of the large variations in blood glucose which are routinely observed in clinical practice. The many factors affecting day/day insulin absorption have been reviewed (Binder, C., Acta Pharmacol Toxicol (Copnh)(Suppl. 2) 27:1-87, 1969.; Binder, C. et al., Scand J Clin Lab Invest 19:156-63, 1967; Berger, M. et al., Diabetes Care 5:77-91, 1982; Schlichtkrull, J., et al., Acta Paediatr Scan (Suppl.) 270:97-102, 1977. Because of the combined effect of the low therapeutic index and the unavoidable variations in daily dosage, insulin therapy must be approached conservatively.
Having to approach insulin therapy conservatively makes it nearly impossible to control blood glucose within the normal range. The result is that the control of glucose and other metabolites in insulin-dependant diabetics is usually far from normal. The great weight of scientific evidence suggests that this poor glucose control is responsible for many if not all of the debilitating and potentially fatal complications of the disease. At onset the average further life expectancy of an insulin-dependant diabetic remains at 35 years, as it was some 71 years ago when insulin was discovered. The production and use of an insulin in which day to day fluctuations in absorption rate have a lesser impact on blood glucose will thus be of great benefit in the treatment and control of diabetes mellitus.
The use of certain phosphorylated insulin produces superior blood glucose control, at least in part, because a given % variation in subcutaneous absorption of the phosphorylated insulin produces a significantly lower change in blood glucose than presently available insulin.
Insulin has previously been phosphorylated by methods employing phosphoric acid (Ferrel R. E. et al., Journal of the American Chemical Society, 70, 2107-7, 1948) or phosphoric acid/POCL.sub.3 in non-aqueous organic solvents using coupling agents (Cerami A. et al., U.S. Pat. Nos. 4,534,894 and 4,705,845) or with phosphoramidate (Rathlev, V. and Rosenberg T., Archives of Biochemistry and Biophysics, 65, 319-339, 1956). The phosphorylated insulin produced by Ferrel et el. and by Rathlev and Rosenberg were part of studies designed to further understand the process of phosphorylation and in particular to increase the knowledge of how it relates to biological systems. No clinical advantage of this phosphorylated insulin was observed.
The patents granted to Cerami et el. involve the production of sulfated and phosphorylated insulin that have the advantage of not polymerizing when stored long-term in insulin delivery systems. Thus, these insulins, as described by Cerami et al. have the advantage of not plugging insulin pumps and, accordingly, for the low percentage of patients using insulin pumps these insulins should produce better control of blood glucose. However, the above insulins did not exhibit physiological properties that would inherently provide better control of blood glucose, to be discussed, and thus there is no claim made to this effect.
Insulin has also been phosphorylated with POCl.sub.3 with excess pyridine as disclosed by Z. Roubal et al., Chemical Abstracts, vol. 68, 1968, (Columbus, Ohio, U.S.). This reference discloses that insulin may be phosphorylated in anhydrous media with essentially no alteration of its hypoglycemic effect.
With respect to differences from the process described herein, the Cerami at al. patents emphasize that the improvement in the process is attained by conducting the phosphorylation in a non-aqueous solvent. Cerami et al. point out in (column 1, lines 39-51 of U.S. Pat. No. 4,705,845) that aqueous conditions are harsh and lead to the destruction of insulin. Accordingly, they teach that the use of sulfuric acid or phosphoric acid and a dehydrating agent in a non-aqueous apolar organic solvent effectively modifies insulin in a non-destructive manner. The process of the present invention described hereinbelow in distinction, is 1) conducted in an aqueous solvent, and 2) conducted under conditions of pH which are not harsh, and 3) produces a product which by process or by purification contains phosphorylated insulin of substantially reduced iso-electric points and which contains substantially no unreacted insulin as did the Cerami et al products (see Tables 1, 2, column 4 of U.S. Pat. No. 4,705,845). With respect to differences in product, the Cerami et al. patents claim phosphorylation only on the free hydroxyl groups of insulin (column 2, lines 29-31 of U.S. Pat. No. 4,705,845). In in the product of the present invention, the predominant phosphorylation is on the free amine groups as well as the tyrosine-OH groups and on the hydroxyl groups of serine and threenine residues.
The present invention thus relates to a product and process not only different from all known prior art, but to one in which the phosphorylated insulin so produced gives superior control of blood glucose due to significantly different pharmicokinetics. This superior ability to control blood glucose when injected subcutaneously has not been observed in any of the prior art relating to phosphorylated insulin. It is believed the improved ability to control blood glucose is at least in part due to a decreased change in blood glucose per % change in insulin dose as compared to unmodified insulin.